There are numerous commercially available systems and/or devices for diagnostic testing of analytes in tissue samples. Currently, a growing number of manufacturers sell single-use lateral flow devices, or test strips, for glucose monitoring. These test strips are part of a segment of in vitro diagnostics called Point-of-Care (POC) devices. Test strips are typically read by either reflectance or electrochemical POC measurement systems.
Reflectant-based glucose strips are composed of three distinct layers; an absorbent pad, a reagent-impregnated membrane, and an enzymatic membrane. Reflectance photometric systems quantitate glucose concentrations by measuring the amount of light reflected from the reagent membrane surface.
Electrochemical systems utilize amperometry or coulometry to quantitate glucose amounts. For amperometric systems, a constant potential is applied to the electrodes and the current flowing through the electrochemical cell is measured. For coulometric systems, the amount of electricity passing between the electrodes is proportional to the quantity of substance manufactured or used by the reduction-oxidation process. In order for POC devices to achieve glucose agreement with central laboratory analyzers, it is often necessary to employ a whole blood control or one that provides close mimicry to whole blood.
Unlike remotely located central laboratories, POC measurement devices provide immediate analyte concentration. These test strips are designed to accept and test whole blood specimens for immediate results. Often such instantaneous glucose level determination is vital for proper diabetes management.
In contrast to POC devices, the methods and “wet” chemistry analyzers used by central laboratories are often less sensitive than POC systems to the “matrix” of the specimen. A “matrix-effect” results when other constituents of a sample besides the analyte tested for produce a matrix shift, or a measurement bias, thereby reducing the accuracy of the analyzer to correctly measure the analyte concentration. Matrix-effects caused by the interaction of processed material and the analytical system may generate an underestimation, or overestimation, of true glucose levels leading to possible misdiagnosis in the patient.
Thus, matrix effects are especially troublesome with Quality Control and Proficiency Testing materials as well as for POC glucose strip manufacturers. The matrix-effect makes it extremely difficult to achieve agreement between the various POC glucose strip systems using a stabilized blood material. Test strips are made by a complicated process of assembling different laminates and/or substrates for removal of cellular components, red blood cells (RBCs) or erythrocytes, enzymes for metabolizing the analyte and reagents to signal a reaction detecting the presence or absence of analyte. By the very nature of the process, significant lot-to-lot variability can occur.
Currently, providers of Glucose Measurement Proficiency Programs must use stabilized materials that exhibit significant matrix effects. The administrators of these programs thus find it necessary to separate the reported results according to the make and model of the glucose monitoring devices. Ideally, the proficiency material should mimic fresh blood so that a single assay value could be used to evaluate the performance of all systems.
POC glucose test strip/instrument manufacturers have a problem in that each lot or batch of strips can differ from others in response to glucose. Variations in manufacturing conditions and materials accounts for this difference. To account for the variations in response, calibration codes are assigned to each strip lot number. Currently, fresh blood samples are used for this assignment. Due to the rapid deterioration of the blood samples, often the only relatively reliable calibrator and/or control is fresh human blood, frequently obtained from POC strip manufacture employees themselves. Precise control of the glucose level is especially difficult since the RBCs in whole blood continue to metabolize. If left at room temperature, the collected blood samples undergo glycolysis. That is, glycolytic enzymes in blood convert the glucose to pyruvic acid and other products by a series of reactions known as the Embden-Meyerhof pathway. To prepare this calibrator, blood is drawn into an anticoagulant to form a suspension. The suspension is then incubated for a few hours to reduce the glucose level, preferably to 50 mg/dL or less. Glucose is then added to achieve the various levels desired for calibration. To maintain the glucose level for future use as a calibrator and/or control, the blood must be immediately stored on ice. While this method can reduce glycolysis, long-term storage on ice can eventually degrade the sample.
Other methods and procedures have been described in the prior art for preparing stable controls and/or calibrators, especially in glucose monitoring instrumentation. One such method has been to immediately completely remove RBCs from fresh whole blood by centrifugation. Since RBCs can comprise up to 50% of the whole blood, the remaining primary components, mainly plasma, do not adequately mimic whole blood in analytical systems. The RBC component contributes to the viscosity, ionic strength and absorption. Because there is failure to achieve total accordance, matrix-effects result.
In glucose lateral flow devices, the presence of the RBCs in the whole blood affects the regulation of sample flow through the various strip layers. That is, the RBCs physically fill and/or plug the pores of the reagent membrane, thus regulating the flow dynamics to the subsequent layer. Moreover, the ionic strength of the RBCs impacts reaction kinetics that in turns shortens, or extends, the reaction phase (i.e. impact slope and time to reach endpoint).
Therefore, in the absence of either the RBCs or ionic strength, sample migration through the test strip layers is markedly different. Rate of absorption, sample delivery through the reagent membrane and flow rate of the enzymatic reaction is changed. Hence, the creation of a stable, control solution that closely simulates cellular components, function characteristics (i.e. size, density, charge and concentration) while operating universally across different POC glucose monitoring devices and maintaining agreement with results obtained from a central laboratory has been heretofore lacking in the prior art.
In accordance with the instantly disclosed invention, a glucose control will be understood to comprise a stable suspension of red cells and plasma, or components which may be combined to produce said stable suspension, that can be analyzed for glucose over a time period of several weeks, with recovery of a consistent value.
In the past various glycolytic inhibitors have been utilized in blood samples to inhibit glycolysis in a sample before the sample is tested. Some common inhibitors include, sodium fluoride, sodium iodoacetate, sodium oxalate, 2-iodoacetamide, d-mannose, either alone or in combination with an anticoagulant. Although these inhibitors will stabilize the glucose in blood specimens, they are usually identified as “incompatible” and/or “interfering” substances when used in combination with many commercially available POC glucose monitors. Therefore, although they inhibit glucose metabolism they do not provide an acceptable control material. Furthermore, it often takes approximately three hours for these glycolytic inhibitors to become fully effective and stabilize glucose levels. During this three-hour lag time, approximately 0.5 mmol/L of glucose can be consumed.
Some cross-linking aldehydes, for example formaldehyde and glutaraldehyde, have been employed to inhibit glycolysis in blood samples. These aldehydes “cross-link” with cellular surface proteins and amino acids. For the purposes of calibration, cross-linking of cellular membrane proteins is not desirable since the characteristics of the whole blood (RBC) would be changed and possibly impact the behavior of the various glucose monitoring systems. While, glyceraldehyde does react with some amino acids to form Schiff bases, it does not “cross-link” with cellular surface proteins. That is, it is mono-functional. In addition, it is rapidly transported through the red blood cell membranes to quickly inhibit glycolysis.
Thus, it has been discovered by the present inventors that not only does the use of non-cross linking aldehydes stabilize glucose values by inhibiting the glycolytic process, but these aldehydes do not affect the rheology (i.e. flow properties) of the RBCs through testing devices. Therefore, it is possible to make a calibrator comprising at least one non-cross linking aldehyde for calibration with the various methodologies ranging from the analyzer in the central laboratory to the POC monitor.